Method for transfecting bovine somatic cells into inducted pluripotent stem cells by adopting transcription factors

A technology of pluripotent stem cells and transcription factors, applied in botany equipment and methods, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve the problem of increasing workload and difficulty, affecting the clinical application of ESCs cells, etc. problem, to avoid the effect of heterogeneous animal cell contamination

Inactive Publication Date: 2010-05-12
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The application of mouse embryonic fibroblasts in the ESCs culture system directly introduces xenogeneic animal cells, which

Method used

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  • Method for transfecting bovine somatic cells into inducted pluripotent stem cells by adopting transcription factors
  • Method for transfecting bovine somatic cells into inducted pluripotent stem cells by adopting transcription factors
  • Method for transfecting bovine somatic cells into inducted pluripotent stem cells by adopting transcription factors

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Experimental program
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Embodiment 1

[0052] Isolation and cultivation of bovine skin fibroblasts of embodiment 1

[0053] Adult bovine dermal fibroblasts (BDFs) were isolated from the ear margin skin of adult female Holstein cows. After the tissue was cleaned and sterilized, it was cut into small tissue pieces, and cultured in high-sugar DMEM (Gibco company product) containing 15%-20% fetal bovine serum (FBS) (Gibco company product) under conventional culture conditions. The culture solution was changed every 2-3 days. After 7-10 days, fibroblasts moved out from the edge of the tissue block, and after 12-16 days, a large number of fibroblasts were closely arranged around the tissue block. Digested with trypsin and EDTA mixed digestive juice and passaged, a large number of adult bovine skin fibroblasts were obtained, see figure 1 . Newborn bull skin fibroblasts (male newborn bovine dermalfibroblasts, NDFs) come from Holstein breed newborn bull ear margin skin, see figure 2 , the method of isolation and culture...

Embodiment 2

[0054] Example 2 Cloning of key gene transcription factor for bovine maintenance of pluripotency and construction of its retroviral vector

[0055] Genital ridges were isolated from fetuses of 50-60-day-old Holstein cows, and the total RNA was extracted with TRIzol LS Reagent after tissue homogenization, and treated with DNaseI (RNase free) to remove genomic DNA contamination. cDNA was obtained by reverse transcription reaction. Then, using cDNA as a template, the complete sequences of the open reading frames (ORFs) of Oct4, Sox2, c-Myc and Klf4 were amplified by PCR respectively. The primer sequences used for gene cloning are shown in Table 1.

[0056] Table 1 The information of the primers used to amplify the four genes of bovine Oct4, Sox2, c-Myc and Klf4

[0057]

[0058]

[0059] The cycle parameters of Oct4 gene PCR amplification are: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 45 sec, annealing at 60°C for 45 sec, extension at 72°C for 1 min, 35 ...

Embodiment 3

[0065] Packaging and preparation of embodiment 3 retrovirus

[0066] PT67 packaging cells were cultured in DMEM medium according to conventional methods. When the cells reached 70%-80% confluence, liposomes were used to transfect retroviruses into packaging cells PT67 cells for virus packaging to obtain infectious retroviruses. virus particles. 1 hour before transfection, DMEM medium was replaced with serum-free medium, 4-8 μg retroviral plasmid DNA and 10-20 μl liposome were added to 500 μl Opti-MEM-I+GlutaMAX-I (Gibco company product) optimization Gently mix in the transfection solution, incubate at room temperature, then gently mix the diluted plasmid DNA and liposomes, and incubate at room temperature for 20 minutes, then add the mixture dropwise to the PT67 cell culture dish. Cells at 37°C, 5% CO 2 After culturing for 4-6 hours under saturated humidity conditions, the transfection solution was replaced with a new DMEM culture solution containing 15% FBS. After 24 hours...

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Abstract

The invention discloses a method for transfecting bovine somatic cells into inducted pluripotent stem cells (iPSCs) by adopting transcription factors, concretely comprising the following steps: 1) isolation and culture of bovine dermal fibroblasts; 2) cloning of bovine transcription factors maintaining pluripotent genes and construction of retroviral vectors of the transcription factors; 3) retrovirus packaging and preparation; and 4) infection of bovine dermal fibroblasts by retrovirus and acquisition of bovine inducted pluripotent stem cells. The invention first adopts the acellular human amniotic membranes as support to replace the mouse embryonic fibroblasts (MEFs) to maintain in vitro proliferation of the bovine iPSCs and keep the bovine iPSCs in undifferentiated pluripotent states for long time. The method which adopts gene inducing to generate the PSCs not only solves the technical problem that the PSCs of the animals such as cattle, sheep, pigs and other livestock are difficult to obtain but also avoids the problem of heterogeneous animal cell pollution caused by the feeder layer which applies the MEFs as the stem cells.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for transfecting bovine somatic cells into induced pluripotent stem cells by using transcription factors. Background technique [0002] In 2006, Japanese scholar Yamanaka [1] It was reported for the first time that mouse somatic cells were co-transfected with a combination of mouse transcription factors Oct4, Sox2, c-Myc, and Klf4, and mouse induced pluripotent stem cells (iPSCs) were obtained. Subsequently, a worldwide research upsurge of induced pluripotent stem cells was set off. Studies have been made on many aspects such as animal species, types and quantities of transcription factors, combinations of transcription factors, and foreign gene introduction methods. 2007Yamanaka [2] Using the same transcription factor to successfully obtain human iPSCs; in the same year, Yu (Yu Junying) and Thomson [3] et al. also successfully obtained human ...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10
Inventor 陈冬梅吕长荣辛晓玲窦忠英
Owner NORTHWEST A & F UNIV
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